Nuclear receptors (NRs) are a major target of drug discovery. NRs are ligand-dependent transcription factors that possess the ability to directly interact with DNA regulating the transcriptional activity of their target genes. These receptors play essential roles in development, cellular homeostasis and metabolism. Moreover, NRs have been implicated in a wide range of diseases and, as such, have been the focus of drug development efforts for the pharmaceutical industry.
In the newest nomenclature for nuclear receptors, Perixome Proliferator Activated Receptors (PPARs), Nuclear subfamily 1 C (NR1C) comprises three subtypes of mammals PPARs: PPARα (also called NR1C1), PPARβ/δ (also called NR1C2) and PPARγ (also called PPARg, glitazone receptor or NR1C3).
PPARs control the expression of networks of genes involved in adipogenesis, lipid metabolism, inflammation and maintenance of metabolic homeostasis [Barish et al., 2006]. Those nuclear receptors activate transcription by binding to elements of DNA sequences, known as peroxisome proliferator response elements (PPRE), in the form of a heterodimer with retinoid X receptors (known as RXRs).
Similar to typical nuclear receptors, PPARs are comprised of distinct functional domains, including an N-terminal transactivation domain (AF1), a highly conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD) containing a ligand-dependent transactivation function (AF2) [Poulsen et al., 2012]. The DNA-binding C domain, composed of two zinc fingers, binds to the peroxisome proliferator response element (PPRE) in the regulatory region of PPAR target genes.
PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the Activator Protein-1 (AP-1), Nuclear Factor-kappa B (NF-kB), signal transducer and activator of transcription 3 (STAT3) and Nuclear Factor of Activated T-cells (NFAT) signaling pathways [Vanden Berghe et al. 2003].
Peroxisome Proliferator-activated Receptor gamma (PPARg) is of special interest because it is involved in the regulation of adipocyte formation, insulin sensitivity and inflammation [Fievet et al. 2006] [Stienstra et al. 2007] [Tontonoz and Spiegelman, 2008]. This nuclear receptor is expressed in a range of tissues including adipose tissue, skeletal muscle cells, osteoclasts, osteoblasts, several immune-type cells, and in the brain and peripheral nervous system.
It is clear that PPARg is the dominant or “master” regulator of adipogenesis, due to the fact that is both sufficient and necessary for fat cell differentiation. The regulatory regions of a large number of genes that play important roles in lipogenesis and insulin sensitivity contain binding sites for PPARg, including aP2, LPL, adiponectin, and Glut4 [Rosen and MacDougald, 2006]. Therefore, activation of PPARg in adipose tissue impacts whole-body insulin sensitivity.
On the other hand, activation of PPARg exerts anti-inflammatory activities in several cell types by inhibiting the expression of pro-inflammatory genes, thereby reducing the production of cytokines, metalloproteases and acute-phase proteins [Tontonoz and Spiegelman, 2008]. It also acts increasing anti-inflammatory cytokines, and inhibiting inducible nitric oxide synthase (iNOS) expression [Széles et al., 2007].
PPARg has been recognized as playing a fundamentally important role in the immune response through its ability to direct the differentiation of immune cells towards anti-inflammatory phenotypes [Tontonoz and Spiegelman, 2008]. Interestingly, PPARg agonists have shown anti-inflammatory and neuroprotective effects in several experimental models of Parkinson's diseases, amyotrophic lateral sclerosis, multiple sclerosis and stroke, as well as in a few clinical studies [Bernardo and Minghetti, 2008]. Additionally, PPARg must formally be considered a tumor suppressor gene in the genetic sense. It is expressed in a variety of tumor cells, and the activation of PPARg by ligands led to either inhibition of cell proliferation or induction of apoptosis [Tachibana et al., 2008] [Tontonoz and Spiegelman, 2008].
The beneficial effects of PPARg activation can be used for the treatment of several PPARg mediated diseases, as is shown in Table 1. For the purposes of present description PPARg mediated disease means any pathological effect observed which might be due to the alteration of PPARg function in normal non-pathological conditions. This table summarizes the actions of PPARs in inflammatory, cancer diseases and other diseases.
TABLE 1DiseaseEffect of PPARγ and its ligandsAtherosclerosis↓Recruitment of immune cells.↓Migration and proliferation of VSMC.Inflammatory↓IL-β-induced IL-8 and MCP-1 in colonic epithelial cells.bowelModulation of inflammatory response: ↓Th1 and ↑Th2.diseasesImprovement of colitis in mice models.Improvement of colitis in 4/15 patients.Rheumatoid ↑Synoviocyte and chrondrocyte apoptosis.arthritis↓TNFα, IL-1β and COX-2 in rheumatoid synoviocytes.Improvement of arthritis in mouse modelsLiver fibrosis↓HSC activation.↓Kupffer cell activation.Nephropathy↓IL-1β, MCP-1, COX-2, iNOS, proliferation and↑apoptosis in mesangial cells.Improvement of micro-albuminuria in Type IIdiabetic patients and diabetic rats.Psoriasis andImprovement of psoriatic lesions in mouse models andskin woundpatients.healingScleroderma↓Collagen production(SSc)↓Fibroblast proliferation and differentiationInteraction with Wnt pathwayNeuro-↓iNOS, TNFα, IL-1β, IL-6, INFγ, MCP-1 and COX-2 indegenerativeastrocytes and microglia.disorders↓Neuronal apoptosis.↑Differentiation of neural stem cellsCancer↑Apoptosis and ↓proliferation of cancer cells.↓Colitis-related colon cancer in mouse models.
Abbreviations: ↓ inhibition, ↑ stimulation, hepatic stellate cells (HSC), vascular smooth muscle cells (VSMC), monocyte chemoattractant protein-1 (MCP-1), T-helper (Th), tumor necrosis factor-α (TNFα), cyclooxygenase (COX), interferon-gamma (INFγ), inducible nitric oxide synthase (iNOS), intracellular adhesion molecule-1 (ICAM-1) [Adapted from Kostadinova et al., 2005].
Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or (Nrf2), is a transcription factor that in humans is encoded by the NFE2L2 gene. The Nrf2 antioxidant response pathway is the primary cellular defense against the cytotoxic effects of oxidative stress. Among other effects, Nrf2 increases the expression of several antioxidant enzymes.
The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by reactive oxygen species (ROS) and electrophiles. The key signaling proteins within the pathway are the transcription factor Nrf2 that binds together with small Maf proteins to the antioxidant response element (ARE) in the regulatory regions of target genes. Under the basal condition, Nrf2-dependent transcription is repressed by a negative regulador, Keap1 (Kelch ECH associating protein 1). When cells are exposed to oxidative stress, electrophiles, or chemopreventive agents, Nrf2 escapes Keap1-mediated repression and activates antioxidant responsive element (ARE)-dependent gene expression to maintain cellular redox homeostasis.
Since this Nrf2-dependent cellular defense response is able to protect multi-organs or multi-tissues, activation of Nrf2 has been implicated in conferring protection against many human diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, acute and chronic lung injury, autoimmune diseases, and inflammation
Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression [Na and Surh 2013].
Cancer cells survive persistent endogenous oxystress or reactive oxygen species (ROS)-induced cellular stress, and become resistant to certain anticancer agents that exert cytotoxicity through ROS production. Under such conditions, an active Nrf2 pathway could maintain a favorable redox balance in cancer cells by keeping ROS levels within a range that promotes their growth and survival. Sustained accumulation or activation of Nrf2 is speculated to confer on a subset of premalignant or cancerous cells an advantageous environment to proliferate, evade apoptosis, metastasize, and tolerate therapeutic intervention.
Inhibition of Nrf2 overexpression has been known to reverse the phenotypic characteristics of cancer cells, lending support to this supposition [Sporn and Liby, 2012]. Constitutive overactivation of Nrf2 has been observed in numerous types of malignancies, such as squamous cell carcinomas, lung cancer, breast cancer, gallbladder cancer, prostate cancer, renal cancer, ependymomas, ovarian epithelial carcinoma, endometrial cancer, and pancreatic cancer [Na and Surh, 2013]. Cancer patients with a constitutively elevated level of Nrf2 expression in their tumor, in general, show a lower survival rate [Solis et al., 2010]. Therefore, Nrf2 is considered a prognostic molecular marker for determining the status of cancer progression and contributes to both intrinsic and acquired chemoresistance. Thus, this antioxidant transcription factor may also act as a proto-oncogene and enhanced Nrf2 activity promotes formation and chemoresistance of solid cancers [Sporn and Liby, 2012].
CBG-Q (compound I), precursor of CBG-Q chemical derivatives (compounds II to XII) of present invention, exerts an activation effect on PPARg. However, CBG-Q also induces activation (see comparative example 4 and FIG. 4) of Nfr2, which provokes a non-desired side effect as tumors becoming resistant to chemotherapy agents, and a chronic treatment with Nrf2-activators may result in carcinogenesis, as explained above. Therefore, the new CBG derivatives of present invention, offer an alternative treatment for cancer more effective due that the side-effect of induced chemotherapy resistance, observed when CBG was administered in vitro, due to Nrf2 over-expression, is not present.
Among activators of PPARg ligands, the thiazolidindiones (TZDs) are of most clinical importance [Lehmann et al., 1995]. For this reason rosiglitazone and pioglitazone have been largely used so far in the clinical practice. They provide similar effects on glycemic control, as well as a range of similar adverse effects, such as weight gain, fluid retention, and increased risk of hearth failure, which seem to be PPARg mediated. Interestingly, those thiazolines differ on their effect on lipid and cardiovascular safety profile, indicating a PPARg-independent mechanism. Indeed, rosiglitazone was recently withdrawn in Europe and its use has been restricted in USA as a consequence of increased risk of cardiovascular events in type 2 diabetic patients.
Although TZDs are potent PPARg full agonists (PPARg-fa) their mechanism-based side effects have limited the full therapeutic potential of those compounds [Gelman et al., 2007] [Ciudin et al., 2012]. But the physiologic and therapeutic relevance of the PPARg pathway have promoted new studies to develop newer classes of molecules that reduce or eliminate adverse effects [Ahmadian et al., 2013]. Therefore, much progress has been achieved in the discovery and development of selective PPARg modulators (PPARg-m) as safer alternatives to PPARg-fa. The preclinical and clinical findings clearly suggest that selective PPARg-m have the potential to become the next generation of PPARg agonists: effective insulin sensitizers with a superior safety profile to that of PPARg-fa. [Doshi et al. 2010].
In this sense natural and synthetic cannabinoids are considered PPARg-m that alleviates inflammatory process through activation of PPARg. Some examples of cannabinoid-based PPARg-m are ajulemic acid [Liu et al., 2003], [Burstein S. 2005], WIN55212-2 [Sun and Bennett, 2007], 9Δ-THC and CBD [O'Sullivan 2007], and CBG and derivatives [Granja et al., 2012].
The clinical relevance of covalent modification of druggable proteins by small molecules has been extensively debated in the past few years by the pharmaceutical industry and some times covalent modification underlies the activity of successful drugs [Singh et al., 2011]. Nevertheless, there is still a rooted bias against covalent drugs irrespective of the mechanism by which they ultimately bind to biomolecules. Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo, including acute cytotoxicity and immunotoxicity [Bolton et al., 2000]. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS) that can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA [Monks and Jones, 2012]. Although there are numerous examples of quinone-based compounds with therapeutic use the concerns over non-specific toxicity and lack of selectivity, the Michael acceptor motif is rarely introduced by design in drug leads.
One example of quinone-based therapeutic compounds is report in the patent WO2011117429 that describes the synthesis of cannabigerol hydroxy-quinone (also named CBG-Q or VCE-003 in the aforesaid international patent application and, for the purposes of present specification, also called compound I) and its use in diseases and conditions responsive to PPARg modulation. Diseases mentioned in WO/2011/117429 are: atherosclerosis, inflammatory bowel diseases, rheumatoid arthritis, liver fibrosis, nephropathy, psoriasis, skin wound healing, skin regeneration, pancreatitis, gastritis, neurodegenerative disorders, cancer; hypertension, hypertrigliceridemia, hypercholesterolemia, obesity and Type II diabetes. The introduction of a quinone motif in the cannabigerol molecule increases its affinity to bind PPARg and increases its transcriptional activity.
Further research shows that cannabigerol hydroxyquinone (CBG-Q or compound I) also activates the transcription factor Nrf2, a cellular sensor of oxidative/electrophilic stress. Thus, introduction of a quinone motif in cannabigerol results in two independent activities such as those exerted as PPARg agonists and Nrf2 activators.
To improve just PPARg agonistic activity, but without inducing activation of Nrf2, in order to avoid induction of resistance to chemotherapy, present invention has developed a library of novel compounds starting from Cannabigerol hydroxyquinone as a template and surprisingly we have found that specific modifications in position 2 resulted on novel compounds suitable for treating PPARg-related diseases due to their high PPARg agonistic effect lacking electrophilic (Nrf2 activation) and cytotoxic activities.
Those cannabigerol hydroxy-quinone derivatives of present invention are different from the compounds described in WO20011117429, since the modifications in position 2 confers to the compounds of the present invention the capacity to activate to PPARg and to protect from glutamate-induced cytotoxicity. These compounds also shown a remarkable low cytotoxicity in cell lines of neuronal origin compared with CBG-Q (compound I) comprised in the state of the art. In addition derivatives of this compounds show therapeutic efficacy in animal models of diseases (Multiple Sclerosis, Parkinson and Huntington diseases) widely used to evaluate the clinical efficacy of PPARg agonists.